Reisomerization of retinal represents a molecular switch mediating Na uptake and release by a bacterial sodium-pumping rhodopsin.

Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the "O intermediate", performs both the uptake and release of Na. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus. We observed two O intermediates, termed O1 and O2, having distinct chromophore structures. We show O1 displays a distorted 13-cis chromophore, while O2 contains a distorted all-trans structure. This finding indicated that the uptake and release of Na are achieved not by a single O intermediate but by two sequential O intermediates that are toggled via isomerization of the retinal chromophore. These results provide crucial structural insight into the unidirectional Na transport mediated by the chromophore-binding pocket of NaRs.